Field of the Disclosure
This Disclosure Relates to Anti-Siglec-15 Immunoconjugates, Antibodies, Antigen-Binding fragments and uses thereof, in particular in the treatment of acute myeloid leukaemia.
Background of the Disclosure
Acute myeloid leukaemia (AML) is a fatal illness where normal haematopoiesis is replaced with a malignant proliferation of immature blast cells. The symptoms, such as infection, bleeding, and anaemia, are related to this bone marrow failure and if left untreated will lead to the death of the patient. AML affects around 2,000 people a year in the UK, with an increasing incidence as the population ages. Curative treatment is highly intensive, which is arduous for the patient, expensive, and requires prolonged hospital admissions, presenting a significant cost to the national health system. In addition, the use of allogeneic bone marrow transplantation, either up-front or for salvage therapy, represents a further cost in those patients for whom it is appropriate. Despite this, the majority of patients with AML still die from the disease.
Many drugs have been developed to treat AML. In addition to conventional chemotherapy agents, therapeutic antibodies have been developed to selectively target AML blasts. Gemtuzumab ozogamicin (GO), targeting a sialic acid binding immunoglobulin-like Siglec protein, CD33, is approved for patients over 60 years of age (Bross P F et al., Clin. Cancer Res. 7(6):1490-6 (2001)).
CD33 is an antigen found on many AML blasts and GO is a toxin-conjugated antibody that binds CD33. Recently, a randomized trial (AML15) reported a disease-free survival advantage for patients treated with GO, mainly related to a decreased incidence of relapse (Burnett A K et al., J. Clin. Oncol. 29(4):369-77 (2011)). However, approximately 10% cases of AML do not express CD33 and GO is also associated with relatively rare, but potentially life-threatening side effects which include myelosuppression, perhaps because CD33 is expressed on healthy hematopoietic stem cells (Taussig D C et al., Blood 106(13): 4086-92 (2005)). In the AML15 trial this side effect appeared to have been manifested by the fact that patients treated with GO required more platelet transfusions and IV antibiotics. In other studies, treatment with GO has also resulted in hepatotoxicity as a dose limiting side effect (Mulford D., Semin. Hematol. 45(2):104-9 (2008)).
Although the existence of cancer stem cells for all malignancies is a much debated topic, it is widely accepted that they are present in leukaemia. The first evidence for the existence of cancer stem cells was demonstrated in acute myeloid leukaemia. Bonnet and Dick isolated the CD34+CD38− subpopulation of leukemic cells and showed that these cells could repopulate tumors in NOD/SCID mice (Bhatia M. et al., Proc. Natl. Acad. Sci. 94(10):5320-5 (1997)). Recent work also suggested that leukaemia stem cell potential is present in the CD38+ compartment (Taussig D C et al., Blood 112(3): 568-75 (2008)). With a growing knowledge of their biology and unique identity, work is beginning to target leukemic stem cells (Chan W I and Huntly B J, Semin. Oncol. 35(4):326-35 (2008)). In leukaemia therapy, active research is targeting this leukaemic stem cell subpopulation, however no drug is available to date that targets these cells specifically.
Siglecs are a family of immunoglobulin superfamily receptors that bind sialic acids (Varki A and Angata T, Glycobiology 16(1):1R-27R (2006)). They are commonly expressed on immune cells, in particular those immune cells of the myeloid lineage. Most Siglecs are inhibitory, but novel activating Siglecs have been discovered recently, namely Siglec-14, -15 and 16 (Angata T et al., FASEB J. 20:1964-1973 (2006), Angata T et al., Glycobiology 17:838-846 (2007), Cao H et al., Eur. J. Immunol. 38:2303-2315 (2008)). Different Siglecs have varying affinities for distinct linkages of sialic acids, which are nine carbon based sugars found at the periphery of most mammalian cell surfaces. Siglec-15 is a newly described Siglec that is well conserved between species with recognizable orthologues of the human sequence in zebrafish. Initial characterisation of Siglec-15 revealed α-2,6 linked sialic acids as its ligand, binding of which was dependent on an essential arginine residue in Siglec-15's N-terminal V-set immunoglobulin domain (Angata T et al., 2007). Siglec-15 has also been shown to bind an antigen highly expressed in a variety of tumors, sialyl-Tn (Angata T et al., 2007). Siglec-15 is unusual in that it is equipped with both negative and positive signalling motifs. In the transmembrane domain, Siglec-15 can associate with positive signalling adaptor molecules, such as DAP10, DAP12 and Fc receptor common γ chain but, at the same time, Siglec-15 encodes a cytoplasmic ITIM-like motif known as immunoreceptor-tyrosine based switch motif (ITSM) that generally mediates inhibitory signals (Shlapatska L M et al., J Immunol. 166(9):5480-7 (2001)). The only other example of an immune receptor encoding dual signalling motifs is KIR2DL4, which associates with ITAM encoding adaptor Fc receptor common γ chain (Miah S M et al., J. Immunol., 180(5):2922-32 (2008)); Kikuchi-Maki A et al., J. Immunol., 174(7):3859-63 (2005)) and also contains an inhibitory ITIM motif in its own cytoplasmic tail (Faure M and Long E O, J. Immunol., 168(12):6208-14 (2002)).
Interest in Siglecs as targets for treatment of leukaemia is growing. Nguyen et al profiled the majority of the known CD33rSiglecs (a major subfamily of Siglecs) for their expression on AML cells. (Nguyen D H, Exp. Hematol., 34(6):728-35 (2006)) They identified expression profiles of Siglecs characteristic of each major subgroup of AML as well as patient-specific CD33rSiglec finger prints. A customized approach to leukaemia treatment has been proposed where full profiling of a patient's Siglec expression is followed by targeting a combination of Siglecs. The advantage of targeting Siglecs is that most of them exhibit rapid endocytosis. Antibody-induced endocytosis of Siglec-5 and Siglec-9 on the surface of U937 cells have similar half-lives of approximately 100 minutes. However, no Siglec-based therapies have been developed for the treatment of AML.